S.aureus is a major pathogen in the community and hospital settings. Because of emerging antibiotic resistance, there is a need to develop new antimicrobial strategies. We have targeted global regulatory elements as a way of developing novel antimicrobial agents. We have identified a regulatory locus called sarA that is involved in the control of several extracellular and cell wall virulence determinants. The sarA locus is composed of three overlapping transcripts, all encoding the SarA protein (14.5 kDa), the major sarA regulatory molecule. Additionally, we recently purified a 13 kDa protein, designated SarR, that binds to the sarA promoter region to down-regulate SarA expression. In searching the S. aureus genome, we discovered a family of SarA-like proteins. Recent crystal structure of SarR (PNAS in press) revealed a dimeric structure as has been proposed for SarA. The structure reveals that SarR represents a new functional class of the "winged helix" family, accommodating an usually long stretch of DNA (about 27 nt) with multiple bending points, eventuating to bending or shortening of target DNA. Based upon the homology and the structural data, we hypothesize that the SarA family of proteins may have homologous structures with similar DNA-binding motifs. Besides structural studies, another major goal of our proposal is to understand the mechanism of gene activation or repression in the SarA family of proteins, using SarA and SarR as models. As such, we have the following aims: I) crystallization of SarR with sarA promoter fragments; II) analysis of the mode of binding of SarA and SarR to the sarA promoter including mutation analysis to precisely map the DNA binding site; III) mutation analysis of SarR to determine the specific activation (or functional) domain; IV) structural and functional analysis of SarS, another SarA homolog; V) deciphering the mechanism of activation and repression of SarR and SarA by examining its interaction with RNAP. In completing these studies, we will be able to determine if the SarA family of proteins possesses conserved secondary structures and binding domains. The SarA family is known to have both activation and repressive functions. Recognizing that both SarA and SarR bind to the UP element to which RNAP interacts, these studies will determine if activation or inhibition of RNAP, by virtue of protein-protein interactions between transcription factor and RNAP, will provide an explanation for the different modes of action for the SarA family. An understanding of these mechanisms will facilitate the development of novel agents that interfere with the function of the SarA family and the ensuing synthesis of cell wall adhesins and toxins.